Human color vision is initiated by light capture in the trichromatic cone mosaic and the subsequent comparison of these signals across space by post-receptoral retinal circuitry. To probe this process in vivo on a single-cell scale requires knowledge of the topography of the trichromatic mosaic as well as an ability to drive individual photoreceptors in isolation. Near the fovea, these efforts are hampered by the optical imperfections that limit the resolution with which the retina can be visualized and stimulated. We sought to overcome these obstacles by imaging and stimulating the retina with a multi-wavelength adaptive optics scanning laser ophthalmoscope. A combination of selective bleaching and cone-resolved retinal densitometry was used to map the trichromatic mosaic at 1.5° eccentricity in two color-normal subjects, and these maps were compared to single-cone increment thresholds collected over the same cone array under L-cone isolating conditions. A short wavelength background (λ = 470 nm) was used to adapt S- and M-cones selectively, such that only L-cones should detect the cone-sized stimulus (λ = 711 nm). There was 90% agreement between image-based and functional maps of the cone mosaic, but L-cone thresholds were widely distributed. Monte Carlo simulations revealed that L-cone thresholds tended to increase as the number of adjacent M-cones increased. Optical modeling ruled out stimulus blur as the primary source of this elevation, suggesting that increased activity in the M-cones engendered by the adapting field serves to inhibit nearby L-cones, either through cone-to-cone electrical coupling or via feedback from inhibitory retinal interneurons.